Therapeutic use of enkephalinase

ABSTRACT

A method and therapeutic composition for the treatment of pathological disorders associated with endogenous peptides by the administration of enkephalinase or derivatives thereof.

This invention, in part, was made with government support under Grant#HL24136 with the National Institutes of Health and the University ofCalifornia. The Government has certain rights in a part of thisinvention.

This is a continuation of co-pending application Ser. No. 07/366,352,filed Jun. 15, 1989 which is a continuation of Ser. No. 117,779, Nov. 5,1987, which is a continuation of Ser. No. 2,473, Jan 12, 1987, all nowabandoned.

BACKGROUND

The present invention relates to the treatment of pathologicalconditions associated with various endogenous peptides. In particular,the invention relates to the use of enkephalinase (E.C. 3.4.24.11) andnovel forms thereof in such treatment.

Various endogenous peptides have been discovered which appear active invarious physiological systems. For example, two pentapeptides, referredto as enkephalins, were extracted from the brain. The effects ofenkephalins include analgesia, thermoregulation, tranquilization,gastrointestinal function and increasing appetite. Until the presentinvention, the sole physiological activity of enkephalinase was thoughtto be the cleavage of enkephalins in the central nervous system(Schwartz, J. C. et al., Trends Pharmacol Sci. 6: 472-476 [1985]). whichto date is not known to be associated with any pathological disorder.Because such activity would result in an enhancement of pain, scientificresearch has focused on inhibiting enkephalinase. This inventionestablishes therapeutic uses of enkephalinase for the first time.

Another endogenous peptide, angiotensin II, is presumed to be anetiologic agent in the pathological condition of renal hypertension.Bradykinin and kallidin have been associated with other pathologicalconditions such as acute inflammation associated with burns, rheumatoidarthritis, edema, carcinoid syndrome, pancreatitis, migraine headache,reactions after transfusion with plasma products, allergic diseases,endotoxic shock and anaphylactic shock.

Another class of endogenous peptides are the tachykinins which sharesome of the same physiological activity. The tachykinins includesubstance P, eledoisin, neurokinin A and B, physalaemin and kassininSubstance P has been shown to be associated with smooth musclecontraction, neurotransmission, pain, cough, exocrine secretion,vasodilation, increased vascular permeability, increased adherence ofleukocytes to venules, stimulation of polymorphonuclear leukocytes,macrophages, T lymphocytes, and degranulation of mast cells. Endogenouspeptides such as bombesin have been found to be present in endocrinecells in normal lungs (Cutz et al., Experientia 37, 765-767 [1981]),released from carcinoid tumors, and implicated in the associatedcutaneous flushes, telangiectasia, diarrhea and bronchoconstriction.

Bombesin functions as a growth factor for airway epithelial cells(Willey et al., Exp. Cell Res. 153, 245-248 [1984]) and for humansmall-cell lung cancer (Cuttitta, F. et al., Nature 316, 823-826[1985]). Substance P and bombesin, apart from their tumor-associatedeffects, have been shown to contract the pulmonary artery and theairways. In light of the observations of the instant invention theseendogenous peptides may mediate various pathophysiologic statesincluding bronchial asthma and hypoxic pulmonary vasoconstriction. Somepeptides are chemotactic, e.g. eosinophil chemotactic factor, C₃ a andsubstance P. They are generated at the site of inflammation and attractvarious immunological cells including neutrophils to the site. Finally,other peptides such as cholecystokinin, somatostatin, oxytocin andcaerulin have potent effects on various tissues which may give rise toother pathological disorders.

Enkephalinase has been purified from kidney (Kerr, M. A. and Kenny, A.J. Biochem. J. 137: 477-488 [1974], Gafford. J. et al., Biochemistry 22,3265-3271 [1983] and Malfroy. B. and Schwartz, J. C. Life Sci 31,1745-1748 [1982]), intestine (Danielsen, E. M. et al., Biochem. J. 191,545-548 [1980]), pituitary (Orlowski, M. and Wilk, S. Biochemistry 20:4942-4945 [1981]), brain (Relton, J. M. et al., Biochem. J. 215: 755-762[1983]) and lymph nodes (Bowes, M. A. and Kenny, A. J., Biochem. J. 236:801-810 [1986]). Enkephalinase has been detected in many peripheralorgans (Llorens. C. and Schwartz. J. C., Eur. J. Pharmacol. 69, 113-116(1981) and in human neutrophils (Connelly, J. C. et al., Proc. Natl.Acad. Sci.[USA] 82: 8737-8741 [1985]). The distribution of enkephalinasein the brain closely parallels that of the enkephalins (Llorens, C. etal., J. Neurochem. 39: 1081-1089 [1982]). The observations of theinstant invention establish that enkephalinase is also present in thoseperipheral tissues and cells that respond to endogenous peptides.Enkephalinase is a membrane-bound glycoprotein with subunit M_(r) valuesin the range of 87,000 to 94,000. Variation in the M_(r) values isattributed to differences in the extent and pattern of glycosylation.

The substrate specificity of enkephalinase has been studied using theenzyme from rat and human kidney. Malfroy, B. and Schwartz, J. C., J.Biol. Chem. 259: 14365-14370 (1984); Gafford et al., Biochemistry 22:3265-3271 (1983); and Pozsgay, M. et al., Biochemistry 25: 1292-1299(1986). These studies indicate that enkephalinase preferentiallyhydrolyzes peptide bonds comprising the amino group of a hydrophobicresidue, shows a marked preference for short peptides, and is mostefficient when it acts as a dipeptidyl carboxypeptidase releasing acarboxy terminal dipeptide. Enkephalinase, which had been found incerebral synaptic membranes, efficiently cleaves the Gly³ -Phe⁴ amidebond of enkephalins (Malfroy, B. et al., Nature (Lond.) 276: 523-526[1978]). Enkephalinase has also been found to cleave the heptapeptide(Met⁵)enkephalin-Arg⁶ -Phe⁷ (Schwartz, J. C. et al., In ProceedingsInternational Union of Pharmacology 9th Congress of Pharmacology, 3: ed.by J. F. Mitchell et al., 277-283, McMillan Press Ltd., London, [1984])as well as a variety of other neuropeptides, such as cholecystokinin(Zuzel, K. A. et al., Neuroscience 15: 149-158 [1985]), substance P(Horsthemke, B. et al. Biochem. Biophys. Res. Comm. 125: 728-733[1984]), neurotensin (Checler et al., 1983), angiotensin I andangiotensin II (Matsas et al., Biochem J. 223 433 [1984] and Gafford etal., Biochemistry 22: 3265 [1983]), kinins, e.g. bradykinin (Gafford. J.T. et al., Biochemistry 22 3265-3271 [1983]), oxytocin (Johnson et al.,1984), and somatostatin (Mumford, R. A. et al., Proc. Natl. Acad. Sci[USA] 78:6623-6627 [1981]). While enkephalinase is capable ofhydrolyzing many biological peptides in vitro (Kenny, A. J. Trends inBiochem. Sci. 11: 40-42 [1986]), in vivo enkephainase has to date onlybeen implicated in the hydrolysis of endogenous enkephalins whenreleased in brain (Schwartz, J. C. et al., Life Sciences 29: 1715-1740[1981] and Lecomte, J. M. et al., J. Pharmacol Exp. Ther. 237: 937-944[1986]). Although the levels of enkephalinase in blood are normally verylow (Connelly et al., supra) enkephalinase was found to be present inhigh levels in the serum from patients with adult respiratory distresssyndrome (Connelly et al. Supra). Enkephalinase cleaves the chemotactictripeptide fMet-Leu-Phe. Id. It was also observed that neutrophils fromdonors who smoked had enkephalinase activites about twice that ofnonsmokers. Id. Enkephalinase has also been found in high levels in themicrovilli of human placentae (Johnson, A. R. et al., Peptides 5:789-796 [1984]).

The present invention is based on the novel observations that specificinhibitors of enkephalinase, thiorphan, leucine-thiorphan andphosphoramidon, potentiate airway mucus secretion and smooth musclecontraction induced by endogenous peptides, e.g. substance P and othertachykinins, and kinins such as bradykinin. The invention is also basedon the novel observation that in vivo enkephalinase inhibits substanceP-induced increases in vascular permeability. Enkephalinase is known tocleave substance P into two fragments observed to be ineffective instimulating mucus secretion and/or smooth muscle contraction. Theinvention is also based on the observation that enkephalinase digestschemotactic molecules and thus may inhibit the attraction of variousinflammatory cells including neutrophils to the site of injury.

An object of the present invention is to provide a therapeuticcomposition for the treatment of pathological conditions in whichendogenous peptides may be involved. Specifically, enkephalinase may beused as a therapeutic agent to overcome adverse effects of substance Por other neuropeptides. More specifically, enkephalinase may be used toreduce peptide-mediated mucus secretion and bronchoconstriction in theairway consequent to various diseases e.g. asthma, chronic bronchitis,cystic fibrosis, and viral infections. Enkephalinase may also be used asa therapeutic agent in the treatment of various tumors, e.g. carcinoidtumors and small cell carcinoma of the lung. Yet another object of thisinvention is the use of enkephalinase derivatives in the treatment ofvarious pathological disorders mediated by certain endogenous peptides.Other peptide-induced disorders may arise in the gastrointestinal,visual, urinary, circulatory, reproductive systems and joints. Thecytoplasmic and/or transmembrane deleted or substituted enkephalinasemay be used in the treatment of various pathological disorders mediatedby certain endogenous peptides.

SUMMARY OF THE INVENTION

The present invention is based on the novel observations that theeffects of endogenous peptides such as substance P and othertachykinins, and/or bradykinin on mucus secretion and smooth musclecontraction in airways are potentiated by specific enkephalinaseinhibitors. The invention is directed to the administration oftherapeutic compositions comprising enkephalinase or derivatives thereoffor the treatment of certain pathological disorders mediated by variousendogenous peptides, for example. bronchoconstriction, airwayhypersecretion, acute inflammation or hyperimmune responses, systemichypertension, cough, infertility or cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Concentration-dependence of substance P (SP)-induced secretion(mean±SE), measured as release of ³⁵ SO₄ -labeled macromolecules(sulfate flux). Tissues from 6 ferrets were incubated with SP at theconcentrations indicated, and the change in sulfate flux for each tissuewas calculated by subtracting the flux of bound SO₄ for the samplecollected immediately before adding SP from the flux of bound SO₄ forthe sample collected either 15 or 30 min after adding SP, whichever wasgreater. The average change in baseline secretion (B) over the 15 minprior to adding SP is shown for comparison (B). SP stimulated therelease of ³⁵ SO₄ labeled macromolecules in a dose dependent fashion.

FIG. 2(A-B). Effects of fragments of substance P (SP) on sulfate fluxfrom two segments from a ferret. Tissues were incubated in chambers with³⁵ SO₄ on the luminal side, and after 3 h, fragments of SP were added tothe submucosal sides of the chambers FIG. 2A: C-terminal fragment, SP6-11 (10⁻⁵ M) stimulated secretion. FIG. 2B: N-terminal fragment. SP 1-9(10⁻⁵ M) had no significant effect on secretion.

FIG. 3(A-B). Effect of proteinase inhibitors on substance P (SP)-inducedchange in sulfate flux from one tissue from a ferret. FIG. 3A: controltissue, incubated with ³⁵ SO₄ and exposed to SP (10⁻⁶ M). FIG. 3B:tissue pre-treated with the combination of 9 proteinase inhibitorsdescribed in the text (9 INHIB) potentiated the secretory response to SP(10⁻⁶ M).

FIG. 4. Effects of proteinase inhibitors on substance P (SP)-inducedchange in sulfate flux (mean±SE) from tissues from ferret tracheas. Openbars: response to SP (10⁻⁶ M) in control tissues from each group.Hatched bars: response to SP 10⁻⁶ M in tissues pre-treated with 9proteinase inhibitors (9 INHIB; 10 μg/ml), phosphoramidon (10⁻⁵ M;PHOSP), thiorphan (10⁻⁴ M; THIOR), captopril (10⁻⁴ M; CAPTO), teprotide(10⁻⁴ M; TEPRO) or other inhibitors (OTHERS) including leupeptin,aprotonin, bacitracin, bovine serum albumin (each inhibitor, 10 μg/ml),or bestatin (10⁻⁵ M). *: p<0.05. Only phosphoramidon and thiorphan(enkephalinase inhibitors) potentiated the secretogogue effects of SP.

FIG. 5. Effects of increasing concentrations of the enkephalinaseinhibitor thiorphan on substance P (SP)-induced sulfate flux fromtracheal segments from 6 ferrets (mean±SE). Filled bars: increase insulfate flux during the 15 min prior to adding drugs. Stippled bars:increase in sulfate flux after adding thiorphan in the concentrationsindicated. Hatched bars: increase in sulfate flux induced by SP (10⁻⁶M). *: p<0.05; compared to the spontaneous increase in sulfate flux; n=6**: p<0.05; compared to the response to SP in control tissues; n=6.Thiorphan potentiated the SP-induced effects on secretion in adose-dependent fashion.

FIG. 6. Effects of tachykinins and of the enkephalinase inhibitor,phosphoramidon, on release of ³⁵ SO₄ -labeled macromolecules from ferrettracheas. Filled bar: Change in sulfate flux during a 15 min baselineperiod (BL) in the absence of drugs. Open bars: Responses to thetachykinins, substance P (SP), neurokinin A (NK-A), neurokinin B (NK-B),physalaemin (PHYS), eledoisin (ELED), and kassinin (KASS) (each drug,10⁻⁵ M). Hatched bars: Responses to tachykinins after pretreatingtissues with phosphoramidon (10⁻⁵ M). *: p<0.05 compared to baseline **:p<0.05 compared to the response to the same tachykinins in the absenceof phosphoramidon. Phosphoramidon potentiated the secretogogue effectsof each tachykinin.

FIG. 7(A-B). Effects of trypsin on enkephalinase activity and substanceP-induced mucus secretion in 3 ferrets. FIG. 7A: Enkephalinase activityin lung homogenates expressed as the degradation of (³ H-Tyr¹, DAla²,Leu⁵)enkephalin. FIG. 7B: Filled bar: change in mucus secretion during a15 min baseline period in the absence of drugs. Open bar: Response tosubstance P (SP). Stippled bar: Change in mucus secretion after addingtrypsin. Hatched bar: Change in mucus secretion induced by SP in tissuespretreated with trypsin. Data expressed as mean±SEM. Trypsin decreasesenkephalinase activity and potentiates secretion induced by substance P.

FIG. 8. Effects of substance P (SP) and of the enkephalinase inhibitorleu-thiorphan on active tension in isolated segments of ferret trachealsmooth muscle. Results are reported as mean±SEM of 12 ferrets (SP≦10⁻⁶M) or 6 ferrets (SP≧5×10⁻⁶ M). Significant differences fromcorresponding control values are indicated by: *=p<0.05; **=p<0.01;***=p<0.001. Substance P alone (open squares) increased tension, butonly at concentrations of 5×10⁻⁶ M and higher. Leu-thiorphan (soliddiamonds) caused a shift in the dose-response curve to lowerconcentrations of SP.

FIG. 9. Effects of receptor antagonists on active tension produced bysubstance P (10⁻⁶ M) in the presence of the enkephalinase inhibitorleu-thiorphan (10⁻⁵ M) in isolated segments of tracheal smooth muscle inferrets. Each point is the mean±SE of the decreases in tension producedby each antagonist compared to the corresponding control responses to SPplus leu-thiorphan. Significant differences from control values areindicated by: *=<0.05; ***=p<0.01. The muscarinic antagonist, atropine(10⁻⁵ M), the SP antagonist, (DPro²,DTrp⁷,9)SP (10⁻⁵ M) and acombination of both drugs decreased SP plus leu-thiorphan-inducedcontractions significantly; the effect of the SP antagonist was greaterthan that of atropine.

FIG. 10. Effects of substance (SP) plus the enkephalinase inhibitorleu-thiorphan on active tension produced by electrical field stimulation(5 Hz) in isolated segments of tracheal smooth muscle in ferrets. Dataare expressed as percent of control responses to electrical fieldstimulation without added drugs and are reported as mean ±SE (N=10at=10⁻⁵ M; n=4 at 10⁻⁴ M). Significant differences from SP alone or SPplus leu-thiorphan are indicated by: *=p<0.05; ***=p<0.001. Substance Palone augmented contractile responses to electrical field stimulation,and this augmentation was potentiated by leu-thiorphan.

FIG. 11. Effects of the enkephalinase inhibitor leu-thiorphan and of thesubstance P (SP) antagonist, (DPro²,DTrp⁷,9)SP on active tensionproduced by electrical field stimulation (5 Hz) in isolated segments oftracheal smooth muscle in ferrets. Data are expressed as percent ofcontrol responses to electrical field stimulation without added drugsand are reported as mean±SE (n=5). Significant differences from controlvalues are indicated by: ***=p<0.01. Leu-thiorphan augmentedcontractions produced by electrical field stimulation, and thisaugmentation was inhibited by the SP antagonist (10⁻⁵ M).

FIG. 12. Effect of substance P (SP) (triangles), neurokinin A (NK-A)(circles) and neurokinin B (NK-B) (squares), and of the enkephalinaseinhibitor, leu-thiorphan, on active tension in isolated segments offerret tracheal smooth muscle. Data expressed as a percent of responsesto acetylcholine (10⁻³ M). Results are reported as mean±SE of 10 ferrets(10⁻⁶ M) and 5 ferrets (10⁻⁵ M) Significant differences betweencontractions with and without leu-thiorphan (10⁻⁵ M) for each tachykininwere obtained. Leu-thiorphan (solid symbols) caused a shift in thedose-response curves to lower concentrations. Although NK-A was morepotent than NK-B in the absence of leu-thiorphan, there were nosignificant differences in muscle contraction between NK-A and NK-B inthe presence of leu-thiorphan (10⁻⁵ M).

FIG. 13. Effect of increasing concentrations of the enkephalinaseinhibitor leu-thiorphan on active tension induced by substance P (SP),neurokinin A (NK-A), and neurokinin B (NK-B) in isolated segments oftracheal smooth muscle in ferrets. Data expressed as percent ofresponses to acetylcholine (10⁻³ M) and reported as mean±SE (n=5 at 10⁻⁵M; n=3 at 3×10⁻⁵ M) Leu-thiorphan potentiated contractions induced byeach tachykinin in a dose-dependent fashion.

FIG. 14. Effect of substance P (SP), neurokinin A (NK-A), and neurokininB (NK-B) and of the tachykinin receptor antagonist, (DPro²,DTrp⁷,9)SP,on active tension produced by electrical field stimulation (5 Hz) inisolated segments of tracheal smooth muscle in ferrets. Data areexpressed as percent of control responses to electrical fieldstimulation without added drugs. Each tachykinin potentiatedelectrically-induced contraction. SP had the most potent effect. Thetachykinin receptor antagonist inhibited this potentiation.

FIG. 15. Effect of increasing concentrations of bradykinin FIG. 15A orlys-bradykinin (FIG. 15B) and of the enkephalinase inhibitor.leu-thiorphan (10⁻⁵ M) on active tension in isolated segments of ferrettracheal smooth muscle. Data are expressed as a percent of responses toacetylcholine (10⁻³ M). Results reported as mean±SE. Significantdifferences between contractions with and without leu-thiorphan areindicated by *=p<0.05. Bradykinin and lys-bradykinin increased activetension in a dose-dependent fashion. Leu-thiorphan caused a shift inboth dose-response curves to lower concentrations.

FIG. 16. Effect of increasing concentrations of substance P and of theenkephalinase inhibitor leu-thiorphan (10⁻⁵ M), on active tension inisolated longitudinal segments of ileal smooth muscle in ferrets.Substance P alone (open circles) caused increased tension in adose-dependent fashion. The enkephalinase inhibitor, leu-thiorphan(solid circles), potentiated the substance P-induced contractions.

FIG. 17(a)-(b). FIGS. 17(a) and 17(b) hereinafter referred to as FIG.17. Amino acid sequence of rat enkephalinase.

FIG. 18(a)-(b). FIGS. 18(a) and 18(b) hereinafter referred to as FIG.18. Amino acid sequence of human enkephalinase.

DETAILED DESCRIPTION

Enkephalinase preferentially hydrolyzes peptide bonds comprising theamino group of a hydrophobic residue, showing a marked preference forshort peptides. The use of enkephalinase as a therapeutic agent forpathological conditions is established by the observations of theinstant invention using as examples, the airway responses and effects oncapillary permeability resulting from release of endogenous peptides,such as substance P or bradykinin. Enkephalinase, also known as neutralendopeptidase or kidney brush border neutral proteinase (E.C. 3.4.24.11,recommended name of the Enzyme Commission), and derivatives thereof maybe used as a therapeutic agent in the treatment of those variouspathological conditions. The experiments are described in detail below.

Endogenous peptides such as enkephalins, angiotensin I and angiotensinII, cholecystokinin, tachykinins e.g. substance P, neurokinin A or B,physalaemin, eledoisin, kassinin, kinins, e.g. bradykinin,lys-bradykinin (kallidin), other peptides, such as neurotensin,oxytocin, somatostatin, bombesin and chemotactic factors, for exampleeosinophil chemotactic factors, have been implicated in variousphysiological and pathological conditions. Examples of effects arecutaneous flushes, telangiectasia, diarrhea and bronchoconstrictionresulting from the release of various peptides such as, substance P,bradykinin and bombesin from carcinoid tumors.

Circulating angiotensin I is converted by angiotensin-converting enzyme(ACE) to angiotensin-II, a potent vasoconstrictor. Systemic hypertensionmay arise from the effects of angiotensin II. Current therapy forsystemic hypertension includes prevention of the conversion ofangiotensin I to angiotensin II by inhibiting ACE. Cough is a sideeffect of this treatment with ACE inhibitors and is proposed to be dueto the inhibition of breakdown of cough-provoking peptides (e.g.,bradykinin). Enkephalinase degrades angiotensin I and II and thus mayserve as an antihypertensive agent. Enkephalinase, by cleavingcough-provoking peptides such as bradykinin should eliminate cough as aside effect of antihypertensive therapy. Another pathological conditionis tachykinin-mediated mucus hypersecretion from submucosal glands ofthe trachea following irritation of the airway epithelium, for example,by an allergen. In addition to mucus secretion there is likely to occur:inflammation, increased capillary permeability, neutrophil chemotaxis,edema and bronchial smooth muscle contraction (bronchoconstriction). Inthe skin, elevated levels of tachykinins or kinins produce sequelae ofsymptoms broadly referred to as dermatitis, including pain, itching,redness and heat and blistering. Tachykinins released in thegastrointestinal and urinary systems have been implicated in secretione.g. salivation from the parotid gland, smooth muscle contraction of theileum and esophagus, effects on frequency of urination, stimulation ofsecretion of water and electrolytes from the jejunum and pancreaticexocrine secretion. Tachykinins also promote embryonic implantation.

The data set forth in this specification establish that enkephalinasewithin the airway degrades substance P and other endogenous peptides toinactive metabolites. This inactivation is a mechanism for mitigatingthe effects of endogenous peptides on mucus secretion andbronchoconstriction This is based on the observation that thiorphan andphosphoramidon, specific inhibitors of enkephalinase, potentiated thesecretory and contractile responses to substance P and other peptides ina concentration-dependent fashion. The inhibitors of enkephalinase wereshown to have no direct effect on mucus secretion or muscle contraction.The effects of the enkephalinase inhibitors and of exogenous tachykininson mucus secretion were observed when administered to the submucosalsurface of tracheal tissue. Inhibitors of other enzyme systems did notalter endogenous peptide-induced secretion. For example, angiotensinconverting enzyme (ACE) is known to be present in the lung and todegrade substance P. However, specific inhibitors of ACE did notpotentiate substance P-induced secretion or bronchoconstriction.Similarly, inhibitors of serine proteases, proteases which are secretedfrom various cells (including neutrophils and mast cells) did notpotentiate substance P-mediated mucus secretion from tracheal tissue.Leu-thiorphan was also observed to potentiate substance P-induced smoothmuscle contraction of ileal tissue, indicating that enkephalinasepresent in gastrointestinal (ileal) tissue inhibits substance P-inducedeffects. The enkephalinase inhibitor, leu-thiorphan, was also observedto inhibit bradykinin and kallidin (lys-bradykinin) induced smoothmuscle contraction of respiratory tissue. This demonstrates thatendogenous enkephalinase inactivates kinins, as well as tachykinins.Thus enkephalinase appears to have a role in modulating endogenouspeptide mediated mucus secretion and/or smooth muscle contraction in theairway. Enkephalinase injected intravenously was shown to inhibitsubstance P-induced extravasation of dye in rats. This provides directevidence that enkephalinase administered to the body can preventpeptide-induced effects. Administration of enkephalinase or derivativesthereof by aerosol would apply the therapeutic agent locally to thetrachea.

As used herein, enkephalinase or enkephalinase derivatives refers toproteins which are enzymatically active or are immunologicallycross-reactive with enzymatically active enkephalinase. Enzymaticallyfunctional enkephalinase is capable of cleaving the Gly³ -Phe⁴ amidebond of ³ H-(DAla², Leu⁵)enkephalin in an assay as described by Llorenset al. (1982).

Enkephalinase or enkephalinase derivatives may be prepared usingpreviously described methods of purification, see e.g. Malfroy andSchwartz. J. Biol. Chem. 259: 14365-14370 (1984) or by recombinant meansas described in copending U.S. patent application Ser. No. 06/946,566,now abandoned and Ser. No. 07/002,478, now U.S. Pat. No. 4,960,700.

Included within the scope of enkephalinase as that term is used hereinare enkephalinase having native glycosylation and the amino acidsequences of rat and human enkephalinase as set forth in FIGS. 17 or 18,analogous enkephalinases from other animal species such as bovine,porcine and the like, deglycosylated or unglycosylated derivatives ofsuch enkephalinases, amino acid sequence variants of enkephalinase andin vitro-generated covalent derivatives of enkephalinases. All of theseforms of enkephalinase are enzymatically active.

Amino acid sequence variants of enkephalinase fall into one or more ofthree classes: substitutional, insertional or deletional variants. Aminoacid sequence variants are characterized by the predetermined nature ofthe variation, a feature that sets them apart from naturally occurringallelic or interspecies variation of the enkephalinase amino acidsequence. The variants typically exhibit the same qualitative biologicalactivity as the naturally-occurring analogue, although variants also areselected to modify the characteristics of enkephalinase as will be morefully described below.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of about from 1 to 10 amino acid residues;and deletions will range about from 1 to 30 residues. Deletions orinsertions preferably are made in adjacent pairs, i.e. a deletion of 2residues or insertion of 2 residues. Substitutions, deletions,insertions or any combination thereof may be combined to arrive at afinal derivative.

Substitutional variants are those in which at least one residue in theFIG. 17 or 18 sequences has been removed and a different residueinserted in its place. Such substitutions generally are made inaccordance with the following Table 1 when it is desired to finelymodulate the characteristics of enkephalinase.

                  TABLE 1                                                         ______________________________________                                        Original Residue  Exemplary Substitutions                                     ______________________________________                                        Ala               ser                                                         Arg               lys                                                         Asn               gln; his                                                    Asp               glu                                                         Cys               ser                                                                           Gln asn                                                                       Glu asp                                                                       Gly pro                                                                       His asn; gln                                                                  Ile leu; val                                                                  Leu ile; val                                                                  Lys arg; gln; glu                                                             Met leu; ile                                                                  Phe met; leu; tyr                                                             Ser thr                                                                       Thr ser                                                                       Trp tyr                                                     Tyr               trp; phe                                                                      Val ile; leu                                                ______________________________________                                    

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table1, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in enkephalinaseproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain. e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine.

A major class of substitutional or deletional variants are thoseinvolving the transmembrane and/or cytoplasmic regions of enkephalinase.The cytoplasmic domain of enkephalinase is the sequence of amino acidresidues commencing at either of two methionine residues shown in FIG.17 (Met⁻⁸ or Met⁻¹) and continuing for approximately 21-24 additionalresidues. In the rat and human sequence the Pro-Lys-Pro-Lys-Lys-Lysdomain is believed to serve as a stop transfer sequence; theconformational bends introduced by the prolyl residues and theelectropositive character provided by the lysyl residues act togetherwith the transmembrane region described below, to bar transfer ofenkephalinase through the cell membrane.

The transmembrane region of enkephalinase is located in the rat sequenceat about residues 21-44 (where Asp is +1 as shown in FIG. 17), and inthe human sequence at the analogous location. This region is a highlyhydrophobic domain that is the proper size to span the lipid bilayer ofthe cellular membrane. It is believed to function in concert with thecytoplasmic domain to anchor enkephalinase in the cell membrane.

Deletion or substitution of either or both of the cytoplasmic andtransmembrane domains will reduce the lipid affinity of the protein andimprove its water solubility so detergents will not be required tomaintain enkephalinase in aqueous solution. Deletion of the cytoplasmicdomain alone, while retaining the transmembrane sequence, will produceenkephalinase which would be solubilized with detergent but which offerstherapeutic advantages. The cytoplasmic domain-deleted enkephalinasewill be more likely to insert into membranes when administered as atherapeutic agent, thereby targeting its activity to the immediateextracellular milieu in which it is ordinarily active, and would improveits formulation in salve or liposomal compositions containinghydrophobic micelles. Preferably, the cytoplasmic or transmembranedomains are deleted, rather than substituted, to avoid the introductionof potentially immunogenic epitopes.

For use in the invention herein, enkephalinase derivatives thereof maybe formulated into either an injectable or topical preparation.Parenteral formulations are known and are suitable for use in theinvention, preferably for i.m. or i.v. administration. Intramuscular ori.v. therapeutic preparations may preferably include transmembrane andcytoplasmic-transmembrane deleted enkephalinase derivative. Theformulations contain therapeutically effective amounts of enkephalinaseor derivatives thereof, are either sterile liquid solutions, liquidsuspensions or lyophilized versions and optionally contain stabilizersor excipients. Lyophilized compositions are reconstituted with suitablediluents (e.g.) water for injection, saline and the like at a level ofabout from 0.001 mg/kg to 25 mg/kg where the biological activity is 10nanomoles/mg/min when assayed at 25° C. in 50 mM, pH 7.4 HEPES buffercontaining 0.1% Tween 20 using (³ H-DAla², Leu⁵) enkephalin assubstrate. Typically, the lyophilized compositions containingenkephalinase will be administered in a range of from about 0.01 mg/kgto about 20 mg/kg of the treated animal. Preferably, the lyophilizedcompositions containing enkephalinase will be administered in a range offrom about 0.1 mg/kg to about 10 mg/kg of the treated animal.

Enkephalinase is formulated into topical preparations for local therapyby including a therapeutically effective concentration of enkephalinasein a dermatological vehicle. Such topical preparations may preferablyinclude the cytoplasmic domain deleted enkephalinase derivative. Theamount of enkephalinase to be administered, and the enkephalinaseconcentration in the topical formulations, will depend upon the vehicleselected, the clinical condition of the patient, the enkephalinase usedand the stability of enkephalinase in the formulation. Thus, thephysician will necessarily employ the appropriate preparation containingthe appropriate concentration of enkephalinase in the formulation, aswell as the amount of formulation administered depending upon clinicalexperience with the patient in question or with similar patients. Theconcentration of enkephalinase for topical formulations is in the rangeof greater than about from 0.1 mg/ml to about 25 mg/ml. Typically, theconcentration of enkephalinase for topical formulations is in the rangeof greater than about from 1 mg/ml to about 20 mg/ml. Solid dispersionsof enkephalinase as well as solubilized preparations can be used. Thus,the precise concentration to be used in the vehicle will be subject tomodest experimental manipulation in order to optimize the therapeuticresponse. Greater than about 10 mg enkephalinase/100 grams of vehiclemay be useful with 1% w/w hydrogel vehicles in the treatment of skininflammation. Suitable vehicles, in addition to gels, are oil-in-wateror water-in-oil emulsions using mineral oils, petrolatum and the like.

Enkephalinase optionally is administered topically by the use of atransdermal therapeutic system (Barry, 1983, DermatologicalFormulations, p. 181 and literature cited therein). Preferred topicalpreparations would comprise enkephalinase including thecytoplasmic-transmembrane domains. Most preferred topical preparationswould comprise enkephalinase lacking the cytoplasmic-domain. While suchtopical delivery systems have been designed largely for transdermaladministration of low molecular weight drugs, by definition they arecapable of percutaneous delivery. They may be readily adapted toadministration of enkephalinase or derivatives thereof and associatedtherapeutic proteins by appropriate selection of the rate-controllingmicroporous membrane.

Topical preparations of enkephalinase either for systemic or localdelivery may be employed and may contain excipients as described abovefor parenteral administration and other excipients used in a topicalpreparation such as cosolvents, surfactants, oils, humectants,emollients, preservatives, stabilizers and antioxidants. Anypharmacologically acceptable buffer may be used e.g. tris or phosphatebuffers. The topical formulations may also optionally include one ormore agents variously termed enhancers, surfactants, accelerants,adsorption promoters or penetration enhancers, such as an agent forenhancing percutaneous penetration of the enkephalinase or other agents.Such agents should desirably possess some or all of the followingfeatures as would be known to the ordinarily skilled artisan: bepharmacologically inert, non-promotive of body fluid or electrolyteloss, compatible with enkephalinase (non-inactivating), and capable offormulation into creams, gels or other topical delivery systems asdesired.

Enkephalinase may also be administered by aerosol to achieve localizeddelivery to the lungs. This is accomplished by preparing an aqueousaerosol, liposomal preparation or solid particles containingenkephalinase or derivatives thereof. Ordinarily, an aqueous aerosol ismade by formulating an aqueous solution or suspension of enkephalinasetogether with conventional pharmaceutically acceptable carriers andstabilizers. The carriers and stabilizers will vary depending upon therequirements for the particular enkephalinase, but typically includenonionic surfactants (Tweens, Pluronics, or polyethylene glycol),innocuous proteins like serum albumin, sorbitan esters, oleic acid,lecithin, amino acids such as glycine, buffers, salts, sugars or sugaralcohols. The formulations also can include mucolytic agents as well asbronchodilating agents. The formulations will be sterile. Aerosolsgenerally will be prepared from isotonic solutions. The particlesoptionally include normal lung surfactants.

Aerosols may be formed of particles in aqueous or nonaqueous (e.g.,fluorocarbon propellant) suspension. Such particles include, forexample, intramolecular aggregates of enkephalinase or derivativesthereof or liposomal or microcapsular-entrapped enkephalinase orderivatives thereof. The aerosols should be free of lung irritants, i.e.substances which cause acute bronchoconstriction, coughing, pulmonaryedema or tissue destruction. However, nonirritating absorption enhancingagents are suitable for use herein. Sonic nebulizers preferably are usedin preparing aerosols. Sonic nebulizers minimize exposing theenkephalinase or derivatives thereof to shear, which can result indegradation of enkephalinase.

Enkephalinase may be administered systemically, rather than topically,by injection i.m., subcutaneously or into vascular spaces, particularlyinto the joints, e.g. intraarticular injection at a dosage of greaterthan about 1 μg/cc joint fluid/day. The dose will be dependent upon theproperties of the enkephalinase employed, e.g. its activity andbiological half-life, the concentration of enkephalinase in theformulation, the site and rate of dosage, the clinical tolerance of thepatient involved, the pathological condition afflicting the patient andthe like as is well within the skill of the physician.

The enkephalinase of the present invention may be administered insolution. Preferably, enkephalinase lacking the transmembrane domain orthe cytoplasmic and transmembrane domains would be used foradministration in solution. The pH of the solution should be in therange of pH 5 to 9.5. preferably pH 6.5 to 7.5. The enkephalinase orderivatives thereof should be in a solution having a suitablepharmaceutically acceptable buffer such as phosphate, tris(hydroxymethyl) aminomethane-HCl or citrate and the like. Bufferconcentrations should be in the range of 1 to 100 mM. The solution ofenkephalinase may also contain a salt, such as sodium chloride orpotassium chloride in a concentration of 50 to 150 mM. An effectiveamount of a stabilizing agent such as an albumin, a globulin, a gelatin,a protamine or a salt of protamine may also be included and may be addedto a solution containing enkephalinase or to the composition from whichthe solution is prepared.

Systemic administration of enkephalinase is made daily, generally byintramuscular injection, although intravascular infusion is acceptable.Administration may also be intranasal or by other nonparenteral routes.Enkephalinase may also be administered via microspheres, liposomes orother microparticulate delivery systems placed in certain tissuesincluding blood. Topical preparations are applied daily directly to theskin or mucosa and then preferably occluded, i.e. protected byoverlaying a bandage, polyolefin film or other barrier impermeable tothe topical preparation.

The method is illustrated by way of the following examples, which arenot to be construed as limiting the invention.

EXAMPLE 1 Measurement of Substance P

The content of substance P-like immunoreactivity in ferret trachea wasdetermined by mincing the trachea in 10 times the tissue weight of 2Nacetic acid, extracting overnight, centrifuging the tissue fragments,and applying the supernatant to a C-18 column (Sep-Pak; Waters andAssociates) that had been equilibrated with water containing 0.1%trifluoroacetic acid (TFA) and pre-cycled with 0.2 ml of 0.5 μg/mlpoly-L-Lysine (Sigma) in 0.1% TFA. The Sep-Pak was then washedsequentially with a step gradient consisting of 4.0 ml each of 0, 20, 40and 60% methanol in 0.1% TFA in water, with the substance P eluting at60% methanol. The methanol was evaporated under N₂, and the samples werethen lyophilized and reconstituted in assay buffer before being assayed.The assay buffer consisted of 0.5% 2-mercaptoethanol, 0.25% bovine serumalbumin (BSA), 0.03% NaN₃ in 0.05M NaPO₄ buffer at a pH of 7.4. Samplesof medium obtained directly from Ussing chambers and the tissue extractswere assayed using a pre-equilibration radioimmunoassay. Separation offree radiolabeled substance P from antibody-bound radiolabeled substanceP was achieved using Dextran T70-coated charcoal (Pharmacia).

Radiolabeled substance P was made by coupling substance P toBolton-Hunter reagent (Bolton, A. E. and Hunter, W. M., Biochem. J. 133:529-539 [1973]) and then purifying the coupled product using HPLC(Hewlett Packard model 1090). The reaction mixture was applied to a 10μm C-18 reverse phase HPLC column (Waters and Associates) and was elutedisocratically from the column at a flow rate of 1 ml/min using 55%methanol and 0.045% TFA in water. The mono-derivatized substance P wasradioiodinated using chloramine-T (McConahey, P. and Dixon, R., Methodsin Enzymology 70: 210-213 [1980]). The reaction mixture was applied to aSep-Pak C-18 column, and the iodinated Bolton-Hunter derivative ofsubstance P was eluted with 60% methanol.

Rabbits were immunized with a mixture of Freund's complete adjuvant,buffered saline and substance P that had been coupled to purified BSAusing glutaraldehyde. Substance P was conjugated by dissolving 25 mgmonomeric BSA and 5 mg substance P in 1.0 mL of 0.1M NaPO₄ buffer at apH of 6.8, and then, with constant stirring, slowly added 0.1 mL of 0.5%glutaraldehyde in NaPO₄ buffer. The mixture was incubated for at least 2hours, after which time, the reaction was quenched by adding 1.0 mL of0.1 M (NH₃)₂ CO₃. After stirring for an additional 30 minutes, themixture was exhaustively dialyzed against distilled H₂ O, lyophilized,and weighed.

Serum was harvested, lyophilized, and frozen. Serum was reconstituted indistilled water for radioimmunoassays. The substance P antiserum wastested for cross reactivity against other peptides including bradykinin,lysyl-bradykinin, vasoactive intestinal peptide, somatostatin,cholecystokinin-8, gastrin, bombesin and neurotensin (Peninsula Labs,Belmont, Calif.). Additionally, we tested several proteinase inhibitors(see below), the N-terminal fragment of substance P, substance P₁₋₉, andthe C-terminal fragment, substance P₆₋₁₁ (Peninsula Labs).

Ferret tracheal segments contained an average of 0.58±0.230 pmoles ofsubstance P-like immunoreactivity per gm. wet weight (n=3), suggestingthat substance P is present in the airways of ferrets.

EXAMPLE 2 Mucus Secretion from Lung Tissue

Mucus secretion from tracheal segments of 36 ferrets was measured usingmethods described earlier (Borson, D. B. et al., J. Appl. Physiol. 57:457-466 [1984]). Briefly after anesthetizing adult male ferrets weighing1-2 kg with sodium pentobarbital (45-60 mg/kg, ip), the trachea wasremoved and washed in medium 199/Earles HCO₃ bubbled with 95% O₂ - 5%CO₂ at 38° C. Tracheal segments were mounted in plastic Ussing-typechambers connected to perfusion chambers made of siliconized glass (MRAInc., Clearwater, Fla.). After exposing both sides to medium 199, 0.167mCi Na₂.³⁵ SO₄ was added to the submucosal side. At 15 minute intervals,the medium was drained from the luminal side and replaced with freshunlabeled medium. Each sample was placed in dialysis tubing (Spectroporeno. D1615-1; MW cutoff: 12-14,000) and exhaustively dialyzed with therest of the samples from that experiment against at least 6 changes ofdistilled water (4 liters each) containing excess unlabeled SO₄ andsodium azide (10 mg/L) to prevent bacterial degradation of themacromolecules. After dialysis, the bound radioactivity of each samplewas determined using a Beckman Instruments beta counter (model LS 7500).Molecular weight profiles of the materials secreted were determined byfirst desalting samples using a Sephadex G-50 column, then concentratingcarbohydrate-containing molecules on DEAE Sephacel, and determiningapparent molecular size under denaturing conditions (4M guanidine, 10 mM2-mercaptoethanol, 0.1% Triton X-100) using a Sepharose CL-6B columnPrevious studies demonstrated that analysis of samples using dialysisand column chromatography yielded quantitatively similar results (Borsonet al., J. Appl. Physiol. 57:457-466 [1984]).

Characteristics of the response to substance P including the timecourse, concentration dependence, and reproducibility were studied.Tracheal segments from 8 ferrets were incubated in the presence ofradiolabel for 3 hours to which were added different concentrations ofsubstance P (10⁻⁹ M to 10⁻⁵ M; Peninsula Labs, Belmont, Calif.).Reproducibility of the response to substance P was established by adding10⁻⁶ M substance P twice, first after 3 hours, and again after 4 hoursof incubation. All treatments were randomized with regard to the site ofsampling in the trachea to eliminate systematic bias.

After mounting tissues in the chambers and adding radiolabel, the rateof secretion of ³⁵ SO₄ -macromolecules into the luminal side of thetissues increased, and by two to three hours, secretion was increasingin an approximately linear fashion. After three hours of labeling,tissues secreted at an average rate of 111.8±15.6 pmol bound SO₄ /cm²/h. At this time, the average increase in flux of bound SO₄ per sampleinterval was 11.7±2.5 pmol bound SO₄ /cm² /h (10.5% of baseline). Whenadded to the submucosal sides of tissues, substance P increasedsecretion within 15 min, after which time, secretion returned towardsbaseline, and finally reached the values obtained by extending thepre-stimulus baseline forward in time. Gel permeation chromatographyindicated that 85% of the radiolabeled materials secreted in response tosubstance P had molecular weights in excess of 10⁶, suggesting that mostwere likely to be mucins or proteoglycans. Substance P increased flux ofbound SO₄ in a concentration-dependent fashion, with a threshold of 10⁻⁹M and a response to 10⁻⁵ M substance P of 156.4±26.1 pmol bound SO₄ /cm²/h or 155±42% above the pre-stimulus baseline (FIG. 1). The substanceP-induced secretion was significantly greater than the increase due totime alone for all concentrations of substance P greater than 10⁻⁹ M(p<0.05; n=6 each). Furthermore, repeated stimulation caused repeatedresponses. The second response was an average of 71.4±24.2% of the firstresponse (p<0.1; n=4). Removing the substance P from the medium afterthe first stimulus increased the magnitude of the second responseslightly to 91.1±48.3% of the first response, but this was notsignificantly different from the second response of the tissues thatwere continuously exposed to substance P (p<0.2; n=4 ea).

The C-terminal fragment, substance P₆₋₁₁ (10⁻⁵ M) caused a potentrelease of bound SO₄, whereas the N-terminal fragment, substance P₁₋₉,did not (369.4±18.3 vs 33.7±24.7 pmol bound SO₄ /cm² /h; p<0.05; n=4 ea)(e.g., FIG. 2). The response to substance P₆₋₁₁ was significantlygreater than the response to substance P₁₋₁₁ at the same concentration(156.4±26.1 pmol bound SO₄ /cm² /h; p<0.001; n=4). Secretion induced bysubstance P₁₋₉ was not different from the spontaneous change in baselineflux in these tissues due to time alone (-18.4±16.8 pmol bound SO₄ /cm²/h; p>0.1; n=4).

EXAMPLE 3 Effect of Proteinase Inhibitors on Substance P-Induced MucusSecretion

In the next series of experiments, the effects of substance P metabolismon secretory responses to substance P was studied. Different enzymescleave substance P at different sites. The active portion of thepeptide, the C-terminal fragment causes secretory activity.Enkephalinase was shown to cleave substance P to the inactive fragmentsubstance P₁₋₉.

The effects of endogenous proteinases were studied by incubating atleast two tissues from each of 4 ferrets for 30 minutes in medium for2.5 hours and adding a combination of nine (9) proteinase inhibitors tothe submucosal side of each tissue (10 μg/mL each). The inhibitors usedincluded leupeptin, antipain, pepstatin A, thiorphan, substance P₁₋₉(Growcott, J. W. and Tarpey, A. V. Eur. J. Pharm 84: 107-109 [1982]) andthe angiotensin converting enzyme inhibitor, teprotide (Peninsula Labs),aprotonin, BSA, and bacitracin (Sigma). Thirty minutes after addition ofinhibitor, substance P (10⁻⁶ M) was added to the submucosal side. In theabsence of proteinase inhibitors, this concentration of substance Pcaused consistent and submaximal effects. In some of the tissues, thecombination of inhibitors stimulated secretion, and when present, theeffect reached a maximum within 30 min after adding the inhibitors. Themean responses to substance P in the presence of inhibitors was comparedwith the mean responses of two control segments from each of the sameanimals. Subsequent experiments tested the effects of the individualproteinase inhibitors, thiorphan, teprotide, phosphoramidon (Sigma),captopril (Squibb Pharmaceuticals, Inc.), teprotide, bovine serum,albumin, bacitracin, leupeptin, aprotonin, and bestatin (Sigma), aninhibitor of an aminopeptidase in brain that degrades enkephalins(Chaillet, P. et al., Eur. J. Pharmacol. 86: 329-336 [1983]).

The concentration-dependence of enkephalinase inhibition was explored byadding thiorphan in different concentrations to different tissues. Todetermine whether the secretory effect of substance P might be mediatedindirectly via the release of endogenous enkephalins, we tested theeffects of met-enkephalin (Peninsula Labs) on secretory responses.

We calculated the flux of bound SO₄ (^(J) SO₄) from the cpm in thedialyzed samples according to the equation modified from (Corrales, R.J. et al , J. Appl. Physiol. 56: 1076-1082 [1985]): ##EQU1##

Changes in flux due to drugs were calculated by subtracting the fluxduring the 15 min period prior to adding drugs from the flux observedfor that tissue either 15 or 30 min after adding drugs, which ever washigher. Mean fluxes or changes in fluxes for each condition werecompared with each other by one-way analysis of variance. Newman-Keulstest for multiple comparisons was used to determine differences betweengroups (Zar, J. H., "Multiple Comparisons" in Biostatistical Analysis[Prentice Hall, Englewood Cliffs, N.J. 1974]).

When added to the submucosal sides of the tissues, the combination of 9proteinase inhibitors increased the flux of bound SO₄ into the lumenslightly, but not significantly (45.8±28.2 pmol bound SO₄ /cm² /h)compared to the increase due to time alone (12.3±6.2 pmol bound SO₄ /cm²/h). After adding substance P to the submucosal sides of these tissues,the flux of bound SO₄ increased by an average of 383.5±148 pmol boundSO₄ /cm² /h, or 438.6±105.2% of the response to substance P of thecontrol tissues from the same animals (83.7±21.4 pmol bound SO₄ /cm² /h,p<0.05; n=4) (FIGS. 3 and 4). The increased response to substance P wasnot due to additive effects of the inhibitors and substance P becausethe sum of the individual effects, 83.7 pmol bound SO₄ /cm² /h(substance P) plus 45.8 pmol bound SO₄ /cm² /h (inhibitors) was 129.5pmol bound SO₄ /cm² /h, or only 34% of the response to substance P inthe presence of the inhibitors (383.5 pmol bound SO₄ /cm² /h).

Of the individual inhibitors studied, only thiorphan and phosphoramidon,inhibitors of enkephalinase, potentiated substance P-induced secretion(FIGS. 4 and 5). When added to the submucosal side of the tissues at10⁻⁴ M, thiorphan by itself increased the flux of bound SO₄ into thelumen by an average of 57±17.8 pmol bound SO₄ /cm² /h. This wassignificantly greater than the spontaneous increase in flux in the sametissues before adding thiorphan (20.5±6.3 pmol bound SO₄ /cm² /h;p<0.05; n=6). At lower concentrations, thiorphan did not stimulatesecretion significantly. However, thiorphan potentiated the secretoryresponse to substance P in a concentration-dependent fashion, with athreshold of approximately 10⁻⁸ M (FIG. 5). In tissues pretreated withthiorphan (10⁻⁴ M), substance P increased flux of bound SO₄ by anaverage of 268.0±58.0 pmol/cm² /h (502±147% of control; p<0.05; n=6).This average response was significantly greater than that of controltissues from the same animals (79.2±13.0 pmol bound SO₄ /cm² /h; p<0.05;n=6). The increase in substance P-induced secretion was not an additiveeffect of thiorphan and substance P: the sum of the individual effects,79.2 pmol bound SO₄ /cm² /h (substance P) plus 57.0 pmol bound SO₄ /cm²/h (thiorphan), or 136.2 pmol bound SO₄ /cm² /h, was only 51% of theresponse to substance P in the presence of thiorphan (268.0 pmol boundSO₄ /cm² /h). In tissues treated with phosphoramidon (10⁻⁵ M), substanceP (10⁻⁶ M) increased flux by an average of 357±91.9 Pmol bound SO₄ /cm²/h, which was significantly greater than the responses of the controltissues from the same animals (62.3±24.8 pmol bound SO₄ /cm² /h; p<0.05;n=4 ea).

In contrast to inhibitors of enkephalinase, inhibitors of otherproteinases did not potentiate substance P-induced secretion (FIG. 4).Thus, captopril and teprotide, inhibitors of kininase II (angiotensinconverting enzyme), potentiated substance P-induced secretion slightly(by 44±27%, and 49±39%, respectively), but the increases were notsignificantly different from responses of the control tissues from thesame animals (p>0.2; n=4 ea). Similarly, leupeptin, aprotonin,bacitracin, BSA. and bestatin failed to potentiate substance P-inducedsecretion.

Experiments were carried out to investigate whether the secretoryresponse to substance P might be mediated by endogenously-releasedenkephalins. Met-enkephalin did not stimulate secretion significantly.After adding met-enkephalin (10⁻⁴ M) the change in flux of bound SO₄ was6.7±4.2 pmol/cm² /h, which was not different from the change in flux incontrol tissues at the equivalent time of 7.0±1.5 pmol bound SO₄ /cm² /h(p<0.5; n=4). These studies indicate that enkephalinase present inairway tissue inhibits substance P-induced secretion.

EXAMPLE 4 Effects of Enkephalinase Inhibitors on Secretory Responses toTachykinins

In this next series of experiments, the effects of different tachykininsand the enkephalinase inhibitor, phosphoramidon were compared. Tissuesfrom ferret tracheas were mounted in chambers and labeled usingpreviously described methods (Example 2). Tissues were then exposed toone of the tachykinins substance P (SP), neurokinin A (NK-A), neurokininB (NK-B), eledoisin (ELED), physalaemin (PHYS), or kassinin (KASS). Eachtachykinin was administered at 10⁻⁵ M in the absence or presence ofphosphoramidon (10⁻⁵ M, 30 min; Sigma). The release of high molecularweight ³⁵ SO₄ was determined as described above in Example 2. The datais shown in FIG. 6. In the absence of phosphoramidon, most of thetachykinins stimulated secretion with an order of potency;

    substance P>physalaemin=eledoisin=kassinin>neurokinin A.

(p<0.05, n=5 each). Neurokinin B, in the absence of phosphoramidon, wasineffective. However, in tissues pretreated with phosphoramidon,secretory responses to each tachykinin were significantly increased(P<0.05, n=5 each). Furthermore, in the presence of phosphoramidon, theorder of potency was altered:

    Substance P=neurokinin A=physalaemin=eledoisin=kassinin>neurokinin B.

In the absence of enkephalinase inhibition, the effect of substance Pwas greater than that of neurokinin A. After enkephalinase inhibition,the effects of substance P and neurokinin A were the same. These resultssuggest that enkephalinase cleaves neurokinin A with greater efficiencythan it cleaves substance P. They also indicate that enkephalinasepresent in airway tissue inhibits secretion induced by varioustachykinins.

EXAMPLE 5 Localization and Functional Properties of Enkephalinase inAirways

In this series of experiments, the tissue locations of enkephalinasewere determined. Enkephalinase was purified from the ferret kidney tonear homogeneity using published methods (Malfroy and Schwartz, Supra).Enkephalinase activity in membrane fractions derived from the vagusnerve, tracheal epithelium, submucosa, muscle, lungs, and kidneys offerrets was determined. Ferrets were anesthetized, and the vagi,trachea, lungs, and kidneys removed. The tracheal epithelium, submucosa,and muscle were separated from each other by incubating the trachea inCa⁺⁺ -free medium for 15 minutes, after which time, the epithelium waseasily removed. The muscle was then separated from the gland-containingsubmucosa. Each tissue was minced and then homogenized in 50 mM HEPESbuffer (pH 7.4) using a Polytron homogenizer.

Enkephalinase activity was measured by determining the rates of cleavageof (³ H-Tyr¹, DAla², Leu⁵)enkephalin (Research Products International)by membrane fractions or purified enkephalinase. Fifty μl of membranefraction was incubated for 40 minutes at room temperature with 50 μl ofbuffer containing (³ H-Tyr¹, DAla², Leu⁵)enkephalin (20 nM), after whichtime the reaction was quenched by adding 50 μl of 2N HCl.

Seventy-five μl of this solution was applied to columns containingpolystyrene beads (Poropak-Q); the characteristic metabolite, ³H-Tyr-DAla-Gly, eluted with water, and the radioactivity determined. Theprotein concentrations were determined by the Bradford procedure(Bradford, M. M. Analytical Biochem. 72:248-254 [1976]), and results ofenkephalinase activities were expressed as fmoles substrate cleaved permin per mg protein. Results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Localization of Airway Enkephalinase                                          And Interactions with Leu-Thiorphan and Substrates                            Tissues                                                                                   Mus-           Kid-  Epthe-  Sub-                                 mucosa      cle     Lung   ney   Vagus/N.                                                                              lium                                 ______________________________________                                        Enke-    1226    520     540 7419*  614     690                               phalinase                                                                     Activity**                                                                            ±211 ±155 ±70     ±116 ±187                            K.sub.I                                                                       leu-    2.1     8.7     2.8  2.1   2.8     3.3                                thiorphan                                                                     (nM)                                                                          (DAla.sup.2,                                                                          53.2    20.0    30.6 32.4  24.1    52.0                               Leu.sup.5)-                                                                   enkephalin                                                                    (μM)                                                                       Substance                                                                             5.4     5.3     3.2  3.6   2.7     4.1                                P (μM)                                                                     Neurokinin                                                                            6.9     9.7     4.4  5.2   3.0     6.1                                A (μM)                                                                     ______________________________________                                         *Mean of triplicate determination from one experiment.                        **Data expressed as fmoles/min/mg protein.                               

Each tissue cleaved the substrate, with the highest activity in theairway present in the submucosa. Leucine-thiorphan (leu-thiorphan, anenkephalinase inhibitor) inhibited substrate cleavage with affinityconstants (K_(I)) in the nanomolar range for each tissue. In contrastwith leu-thiorphan, other proteinase or peptidase inhibitors (10⁻⁵ M),did not inhibit substrate cleavage by any tissue. These inhibitorsincluded: captopril, an inhibitor of angiotensin converting enzyme;bestatin, an inhibitor of aminopeptidases; aprotonin, an inhibitor ofserine proteinases; or, leupeptin, an inhibitor of serine or thiolproteinases. Therefore, substrate cleavage is due exclusively to theaction of enkephalinase. The peptides, (DAla²,Leu⁵)enkephalin, substanceP, and neurokinin A also inhibited substrate cleavage with affinityconstants (K_(I)) that, for each substrate, were the same for alltissues (see Table 2). The affinity of enkephalinase for substance P andfor neurokinin A is approximately ten times the affinity ofenkephalinase for (DAla²,Leu⁵)enkephalin. Because the affinity constantsfor substance P and for neurokinin A are the same, the differences inactivities of these peptides on mucus secretion (Example 4) and musclecontraction (Example 8) are probably due to differences in turnovernumbers (Kcat) and not to differences in affinities of enkephalinase forthe substrates.

EXAMPLE 6 Effect of Trypsin on Enkephalinase Activity and SubstanceP-induced Mucus Secretion

In this series of experiments, the effects of a prototype extracellularproteinase on enkephalinase and substance P-mediated mucus secretionwere studied. The rationale for these studies is that a variety ofproteinases are released from different cells and tissues (e.g.neutrophil elastase, alkaline proteinase, mast call tryptase andchymase), and if these proteinases destroy enkephalinase, thenalterations in peptide-induced secretion should be observed. Thishypothesis was tested using trypsin (10⁻¹¹ to 10⁻⁵ M; 15 min) aprototype serine proteinase. The effect of trypsin on enkephalinaseactivity of lung homogenates measured as degradation of (³ H-Tyr-DAla²-Leu)enkephalin, and mucus secretion from tracheas of 3 ferrets wasdetermined.

Trypsin incubation (15 minutes) decreased enkephalinase activity inhomogenates of lungs (FIG. 7) in a concentration-dependent fashion, witha threshold above 10⁻¹¹ M and a maximal effect at 10⁻⁵ M. Additionally,trypsin (10⁻⁵ M, 30 min) by itself did not increase mucus secretion morethan the increase due to time alone. However, trypsin increased thesecretory response to substance P. Thus, as is the case forenkephalinase inhibitors (e.g. thiorphan), the decrease in enkephalinaseactivity caused by trypsin is associated with increased secretoryresponses to substance P. Therefore, it is likely that other proteinases(e.g. those from inflammatory cells, mast cells, neutrophils, etc.) alsomight alter peptide-induced responses indirectly, by regulating theamount or activity of enkephalinase in the tissue.

EXAMPLE 7 Effect of Proteinase Inhibitors on Substance P-Induced AirwaySmooth Muscle Contraction

Twenty-seven ferrets were anesthetized with pentobarbital sodium (45-60mg/kg, i.p.), and the trachea were removed. Transverse rings (8 mm long)were cut from the trachea and mounted in glass chambers filled with 14ml of Krebs-Henseleit solution of the following composition (inmMoles/L): NaCl 118, KCl 5.9, CaCl₂ 12 2.5, MgSO₄ 1.2, NaH₂ PO4 1.2,NaHCO₃ 25.5, glucose 5.6, 0.1% bovine serum albumin, andpenicillin-streptomycin (100 Units/ml). The solution was maintained at37° C. and was aerated continuously in a mixture of 95% O₂ -5% CO₂,which produced a pH of 7.4. Six tracheal rings were studiedconcurrently.

The tracheal rings were connected to strain gauges (Grass FT03) forcontinuous recording of isometric tension, and the rings were placedbetween two rectangular platinum electrodes (6×40 mm) for electricalfield stimulation. The rings were initially stretched to a tension of 20g for 30 seconds and were then allowed to equilibrate for 1 hour whileresting tension was adjusted to 4 g (Skoogh, B-E. et al., J. Appl.Physiol. 53: 253-257 [1982]). During equilibration, the medium waschanged every 15 minutes. Preliminary studies showed that maximalresponses to electrical field stimulation (biphasic, pulse duration 0.5ms; 20 V for 20 s, frequency, 20 Hz) were obtained with 4 g of restingtension. Id.

The responsiveness to substance P (using concentrations ranging from10⁻⁸ M to 10⁻⁵ M) and to leu-thiorphan and cumulative dose-responsecurves to substance P (Peninsula Labs) were obtained. Each succeedingconcentration of substance P was added after contraction reached aplateau. After completion of the first dose-response curve to substanceP, leu-thiorphan (10⁻⁵ M, Squibb Pharmaceutical) was added to the organbath. Following a 15 minute incubation, a second dose-response curve tosubstance P was obtained.

The N-terminal fragment, substance P₁₋₉ was studied becauseenkephalinase cleaves substance P between the 9 and 10 positions(Matsas, R. et al., Proc. Natl. Acad. Sci. [USA] 80: 3111-3115 [1983]).generating that fragment. (10⁻⁵ M Peninsula Labs). Experiments were alsocarried out with (DPro², DTrp⁷,9)-substance P (10⁻⁵ M) (Peninsula Labs),a substance P antagonist (Hakanson, R. et al., Br. J. Pharmac. 77:697-700 [1982]) by adding the antagonist after the response to substanceP (10⁻⁶ M) and leu-thiorphan (10⁻⁵ M) reached a plateau.

Substance P was dissolved in 0.1M acetic acid, and leu-thiorphan wasdissolved in 1% ethanol to give stock solutions of approximately 10⁻³ M.These drugs were stored at -25° C., and aliquots were thawed and dilutedin Krebs-Henseleit solution for each experiment.

Data were expressed as mean±SE. For the dose-response curves tosubstance P, the means between two groups at each concentration werecompared by a paired t test. For the studies of electrical fieldstimulation, responses were compared by one way analysis of variance andNewman-Keuls multiple range test. Significance was accepted at P<0.05.

Substance P alone caused an increased muscle tension, but only atconcentrations of 5×10⁻⁶ M or greater (FIG. 8). Addition ofleu-thiorphan (10⁻⁵ M) alone had no significant effect on restingtension, but it shifted the dose-response relationship to substance P tolower concentrations by approximately one log unit (FIG. 8). In contrastto substance P, the N-terminal fragment substance P₁₋₉ (10⁻⁵ M), had nosignificant effect on resting tension (n=3).

The contraction produced by substance P (10⁻⁶ M) in the presence ofleu-thiorphan was decreased by the substance P antagonist (DPro²,DTrp⁷,9)substance P (10⁻⁵ M) (36.0±10.0% of control). The substance Pantagonist decreased substance P-induced contractions significantly morethan did atropine alone (p<0.01) (FIG. 9).

Electrical field stimulation-induced contraction in the presence ofsubstance P (5×10⁻¹¹ M) and either captopril (Squibb Pharmaceutical)(10⁻⁵ M), bestatin (Sigma) (10⁻⁵ M) or leupeptin (Peninsula Labs) (10⁻⁵M) was measured to determine whether inhibition of angiotensinconverting enzyme (ACE), aminopeptidases, and serine or thiolproteinases were responsible for potentiating responses to electricalfield stimulation. Increasing concentrations of leu-thiorphan (10⁻¹¹ to10⁻⁴ M) were added and the stimuli repeated to determine whetherleu-thiorphan modulated electrical field stimulation-inducedcontractions. The effects of leu-thiorphan on electrically inducedcontraction was studied using five additional ferrets. After determiningcontrol responses to electrical field stimulation (5 Hz), leu-thiorphan(10⁻⁵ M; 15 min) was added, the stimulus repeated,(DPro²,DTrp⁷,9)substance P, the substance P antagonist (10⁻⁵ M; 15 min;Peninsula Labs) was added, and the stimulus was repeated.

Substance P alone, even in very low concentrations (5×10⁻¹¹ M),augmented the responses to electrical field stimulation (FIG. 10).Substance P (10⁻¹¹ or 10⁻¹⁰ M), reproducibly augmented electrical fieldstimulation-induced contraction, with no significant tachyphylaxis ofthe effect, even after 5 responses (n=3). Leu-thiorphan atconcentrations up to 10⁻⁴ M did not alter resting tension in anyexperiment. However, addition of increasing concentrations ofleu-thiorphan produced dose-related increases in the responses toelectrical field stimulation, with a threshold of approximately 10⁻⁹ Mand a maximum effect at 10⁻⁵ M (FIG. 10).

Leu-thiorphan potentiated the response to electrical field stimulationin a concentration-dependent fashion (FIG. 11). (DPro²,DTrp⁷,9)substanceP (10⁻⁵ M) significantly inhibited the increase in the response toelectrical field stimulation induced by leu-thiorphan (101.6±1.7%compared to 118±1.7% in the presence of leu-thiorphan; p<0.01; n=5).

In contrast to the effects of leu-thiorphan, none of the other peptidaseinhibitors used potentiated the response to electrical field stimulationin the presence of substance P (5×10⁻¹¹ M) (p<0.5; n=3).

These studies establish that enkephalinase is present in airway muscleand nerves, and that it decreases substance P-induced effects, includingbronchoconstriction and potentiation of neurotransmission to airwaysmooth muscle.

Enkephalinase is involved in decreasing substance P-induced effectsbecause leu-thiorphan augmented substance P-induced effects, whereasother proteinase and peptidase inhibitors such as captopril, aninhibitor of angiotensin converting enzyme (Turner. A. J. et al.,Biochem. Pharmacol. 34: 1347-1356 [1985]), bestatin, an inhibitor ofaminopeptidases, or leupeptin, an inhibitor of serine or thiolproteinases, were without effect.

EXAMPLE 8 Effect of Proteinase Inhibitors on Tachykinin InducedContraction of Airway Smooth Muscle

The methods used to excise tracheal tissue, mount and electricallystimulate the tissue were identical to those described above. Theresponses to tachykinins and to leu-thiorphan, cumulative dose-responsecurves to substance P (Peninsula Labs), neurokinin A (Peninsula Labs)and neurokinin B (Peninsula Labs) were obtained using concentrationsranging from 10⁻¹¹ M to 10⁻⁵ M in the presence and absence ofleu-thiorphan (10⁻⁵ M, 15 min; Squibb Pharmaceutical). Each succeedingconcentration of tachykinin was added after the previous contraction hadreached a plateau.

Electrical field stimulation-induced contractions were measured in thepresence of substance P (10⁻¹⁰ M), neurokinin A (10⁻¹⁰ M) and neurokininB (10⁻¹⁰ M), combined with either captopril (10⁻⁵ M; 15 min; SquibbPharmaceutical), bestatin (10⁻⁵ M); 15 min; Sigma) or leupeptin (10⁻⁵ M;15 min; Peninsula Labs) to determine whether inhibition of angiotensinconverting enzyme (ACE), aminopeptidases, or serine or thiol proteinaseswere responsible for potentiating responses to electrical fieldstimulation.

Data were expressed as mean±SE. For the dose-response curves totachykinins the means between two groups at each concentration wereanalyzed by an unpaired t test. For the study of electrical fieldstimulation, responses were compared by one way analysis of variance andNewman-Keuls multiple range test. Significance was accepted at p<0.05.

Substance P, neurokinin A and neurokinin B alone caused smooth musclecontraction, but only at concentrations of 10⁻⁶ M or greater forneurokinin A and 10⁻⁵ M for substance P and neurokinin B (FIG. 12).Contractions induced by tachykinins were compared at 10⁻⁶ M and 10⁻⁵ M,indicating a rank order of potency:

    neurokinin A>substance P>neurokinin B,

although statistically significant differences were obtained onlybetween neurokinin A and substance P (p<0.05) or neurokinin A andneurokinin B (p<0.01) at 10⁻⁶ M, and between neurokinin A and neurokininB (p<0.01) at 10⁻⁵ M. Addition of leu-thiorphan (10⁻⁵ M) alone had nosignificant effect on resting tension, but shifted the dose-responsecurves to substance P, neurokinin A and neurokinin B to lowerconcentrations by approximately one-log unit for substance P, two-logunits for neurokinin A, and three-log units for neurokinin B (FIG. 12).A rank order of potency of tachykinins in the presence of leu-thiorphanwas:

    neurokinin A=neurokinin B>substance P.

Leu-thiorphan potentiated the responses to tachykinins in aconcentration-dependent fashion, with a threshold of approximately 10⁻⁷M and a maximum effect at 10⁻⁵ M (FIG. 13). In contrast to the effectsof leu-thiorphan, none of the other peptidase inhibitors usedpotentiated the response to electrical field stimulation in the presenceof substance P, neurokinin A or neurokinin B (10⁻⁷ M).

Substance P, neurokinin A and neurokinin B each potentiated theresponses to electrical field stimulation in a concentration-dependentfashion. Substance P potentiated the response to electrical fieldstimulation significantly more than

EXAMPLE 9 Effects of Enkephalinase Inhibitors on Bradykinin InducedAirway Smooth Muscle Contraction

The methods used were the same as those described in Example 7.Bradykinin (Sigma) and lys-bradykinin (kallidin; Sigma) causedcontraction in a dose-dependent fashion (10⁻¹¹ to 10⁻⁵ M) (FIG. 15). Inthe absence of enkephalinase inhibitors, bradykinin was more potent thanlys-bradykinin. Leu-thiorphan (10⁻⁶ M) shifted the dose-response curvesto lower concentrations by 1 to 1.5 log units. In the presence ofLeu-thiorphan, the contractile effects of bradykinin and lys-bradykininwere equally potent. This study demonstrates that enkephalinase presentin airway tissue decreases the effect of kinin-induced smooth musclecontraction.

EXAMPLE 10 Effects of Enkephalinase Inhibitors on Substance P-InducedIleal Smooth Muscle Contraction

Using methods similar to those with ferret airway smooth muscle,described in Example 7, longitudinal ileal smooth muscle was examined.In the control state, in the absence of enkephalinase inhibitors,substance P caused tonic contraction in a dose dependent fashion (FIG.16). Leu-thiorphan (10⁻⁵ M) shifted the dose response curve to lowerconcentrations by 1 log unit. This study demonstrates that enkephalinasepresent in gastrointestinal (ileal) tissue decreases the effect ofsubstance P-induced smooth muscle contraction. neurokinin A andneurokinin B. (DPro², DTrp⁷⁻⁹)substance P (10⁻⁵ M, a specific tachykininantagonist) inhibited substance P-, and neurokinin A-, and neurokininB-induced potentiating responses to electrical field stimulation (FIG.14).

These studies establish that substance P, neurokinin A and neurokinin Bcause smooth muscle contraction and they potentiate neurotransmission toairway smooth muscle with different potencies. Enkephalinase present inairways is an important inhibitor of tachykinin-induced effects. Themechanism by which leu-thiorphan potentiates tachykinin-induced effectsis most likely by preventing degradation of the peptides byenkephalinase. The sensitivity to hydrolysis of different tachykinins byenkephalinase may be the explanation of the change in rank order ofpotency in tachykinin-induced effects.

The substance P antagonist, (DPro², DTrp⁷,9)substance P inhibitedelectrical field stimulation-induced contraction. suggesting that theeffects of tachykinins are mediated via tachykinin receptors becausethis antagonist is selective for tachykinins (Leander, S. R. et al.,Nature 294: 467-469 [1981]).

As shown above, the tachykinin-induced effects in ferret trachea may bemediated by enkephalinase because leu-thiorphan augmentedtachykinin-induced effects, whereas other inhibitors of proteinases andpeptidases did not. Those inhibitors described above include: captopril,an inhibitor of angiotensin converting enzyme; bestatin, an inhibitor ofaminopeptidases; and leupeptin, an inhibitor of serine or thiolproteinases. Furthermore, the effects of leu-thiorphan ontachykinin-induced contractions were concentration-dependent (FIG. 13).suggesting that activity of endogenous enkephalinase is closely relatedto tachykinin-induced effects.

EXAMPLE 11 Chemotactic Assay

The normal functions of mature neutrophils are chemotaxis, phagocytosis,microbicidal action, and digestion of foreign material. Chemotacticfactors are generated at the site of inflammation which attract variousimmunological cells including neutrophils to that site. The mechanismunderlying the chemotactic attraction of neutrophils to the inflammatorysite is not fully understood. Enkephalinase has been implicated in themechanism. Connelly, J. C. et al., Proc. Natl. Acad. Sci. (USA) 82,8737-8741 (1985). In certain cases of hyperimmune responses abnormalinflux of neutrophils and other immune cells may cause additional tissuedamage.

Enkephalinase has been found to be bound to the cell membrane of humanneutrophils. Connelly, et al., supra. Membrane bound enkephalinase fromneutrophils cleaves the chemotactic peptide fMet-Leu-Phe (Id.)Neutrophil degranulation and chemotaxis require cleavage of chemotacticpeptides (Smith, R. et al., Fed. Proc. Fed. Am. Soc. Exp. Biol. 44, 576[1985]) and Aswanikumar, S. et al., Proc. Natl. Acad. Sci. (USA) 73,2439-2442 [1976]). Thus, it has been suggested that neutrophil membranebound enkephalinase may be associated with the chemotactic signal bycleaving fMet-Leu Phe in the immediate vicinity of the neutrophilreceptor. This degradation would control the local concentration of thechemotactic peptide.

An assay was used to test the effects of enkephalinase on neutrophilchemotaxis. See U.S. patent application Ser. No. 06/707,005 now U.S.Pat. No. 4,714,674. Neutrophils were isolated by sedimentation overdextran from peripheral blood of human donors. A sample of neutrophilsis placed over a 5 μm filter in a chemotaxis chamber containing aliquotsof test material. Three to six replicates were run for each test for 1hr. at 37° C. The number of migrating neutrophils in each chamber isthen counted. The chemotactic potential is evaluated by the number ofcells in five selected unit areas. A commercially available chemotaxiskit, Neuroprobe, Cabin John, Md. was used. Various specific inhibitorsof enkephalinase were used to determine the role of enkephalinase inchemotaxis of neutrophils. Chemotactic activity is reported as the totalnumber of neutrophils observed in five fields of the kit membrane under100× magnification. Thus, the larger the number the more chemotactic wasa particular test composition.

                  TABLE 3                                                         ______________________________________                                        Chemotactic Activity                                                                            Neutrophil Migration                                                          (% control)                                                 ______________________________________                                        Formyl Met--Leu--Phe 1 μM                                                                      100                                                       Formyl Met--Leu--Phe +                                                                            29 ± 19 (n = 5)                                        Thiorphan 10 μM                                                            Formyl Met--Leu--Phe +                                                                            65 (n = 1)                                                Phosphoramidon 10 μM                                                       ______________________________________                                    

Thus, neutrophil enkephalinase modulates chemotactic activity.

EXAMPLE 11 Enkephalinase Cleavage of Bombesin

Bombesin and bombesin-like peptides have been shown to function asgrowth factors for airway epithelial cells (Willey et al., supra) and inhuman small-cell lung cancer (SCLC) (Cuttitta et al., Nature 316:823-826 [1985]). It was shown that a monoclonal antibody to bombesininhibited the in vivo growth of SCLC cells in mice. Id.

Bombesin (10⁻⁴ M) and (Leu⁵)enkephalin (10⁻⁴ M) were added to purifiedrat kidney enkephalinase (50 ng in 150 ul 50 mM, pH7.4 HEPES buffercontaining 0.02% Triton X-100). After 30 minutes at 37° C., 50 μl 2N HClwas added and the incubation medium was analyzed by HPLC (C18 μBondapakcolumn, 30 minute linear gradient from 0 to 75% acetonitrite in 0.1%trifluoroacetic acid). While 15% of the (Leu⁵) enkephalin was founddegraded, as much as 80% of the bombesin was hydrolyzed. Bombesinappears to be a good substrate for enkephalinase. Administration oftherapeutically effective amounts of enkephalinase may retard the growthof a tumor requiring bombesin for cellular proliferation.

EXAMPLE 13 In Vivo Studies of the Effects of Enkephalinase on SubstanceP-Induced Extravasation

This study demonstrates that substance P injection increases theextravasation of Evans Blue dye. Pretreatment of animals withenkephalinase reduced those effects.

Male Long Evans rats were used for this study. Each rat (250 gm) wasanesthetized by injecting sodium methylhexabarbital (75 mg/kg, ip), andplaced in a supine position. Venous cutdowns in the jugular or femoralveins were made for intravascular injections.

Extravasation of Evans Blue dye was measured to study changes invascular permeability. After intravascular injection, the dye mixesrapidly in the vascular system where it binds to serum albumin, therebycreating a high molecular weight dye-protein complex. The dye-proteincomplex remains in the vascular system unless blood vessel permeabilitywas increased. Permeability changes were monitored by injecting EvansBlue dye solution (0.250 ml of a 30 mg/ml solution in saline) into thevenous circulation. After 1 minute, substance P (1 μg/kg) in saline wasinjected. Five minutes later, the animal was perfused with a fixativesolution consisting of 0.05M citrate buffer (pH 3.5) containing 1%paraformaldehyde for 2 minutes. The acidic fixative prevents the EvansBlue from diffusing out of the tissues. After fixation, the skin of thefeet and nose was dissected free from the underlying connective tissue,weighed, and placed in 2.0 ml of formamide (50° C., 24 hours) to extractthe dye. The amount of dye extracted from each tissue was determined bymeasuring the absorbance at 620 nm and comparing the results with astandard curve for known concentrations of dye. The Evans Blue contentis expressed as ng dye/gm tissue wet weight.

Preliminary experiments were designed to determine the dose of substanceP that caused reproducible, modest responses. Experiments showed that 1μg/kg was a satisfactory does. Subsequent experiments were designed todetermine whether enkephalinase inhibits responses to substance P.

Enkephalinase was purified from rat kidneys using published methods(Malfroy and Schwartz, J. Biol. Chem. [1984]) and was concentrated to 1mg/ml buffer for use. The buffer consisted of 5 mM HEPES (pH 7.4)containing 0.1% Triton X-100 and 500 mM methyl-α-D-glucopyranoside. Theeffect of enkephalinase on the responses to substance P was studied byinjection of the enzyme (100 μg i.v.) per animal 15 minutes prior toinjection substance P.

Results of these experiments are shown in Table 4. These studiesdemonstrate that substance P increased vascular permeability in the skinof the nose and paws. Studies showed that 1.0 μg/kg of substance P(intravenous) increased the permeability to Evans Blue dye, and thatenkephalinase (100 μg) inhibited the vascular responses in the skin inthree of four experiments to substance P.

                  TABLE 4                                                         ______________________________________                                        Effect of Enkephalinase on Vascular Permeability                              Experiment                                                                    number     Control    Enkephalinase                                                                             % Control                                   ______________________________________                                        Nose                                                                          Experiment #R-4                                                                           18.3 ± 1.12*                                                                         12.1        66.2                                        Experiment #R-6                                                                          58.4       30.0        51.3                                        Feet                                                                          Experiment #R-4                                                                          9.4 ± 2.7                                                                             11.1        118                                         Experiment #R-6                                                                          11.9       3.6         29.9                                        ______________________________________                                         *Evans Blue Content in ng/gm wet weight                                  

EXAMPLE 14 In Vivo Effects of Enkephalinase on Airflow Resistance

The effects of enkephalinase on airway smooth muscle in vivo, is studiedusing previously published methods (Holtzman et al. Am. Rev. Respirat.Disease 127: 686 [1983]). Airflow resistance is measured in anesthetizedanimals (sodium pentobarbital, 30 mg/kg, ip, or chloralose, 40 to 60mg/kg, iv). An endotracheal tube is inserted into the upper trachea andthe animal is ventilated according to its size using a constant volumeventilator. Large animals such as dogs require tidal volumes of 10 ml/kgat a frequency of 30 breaths per minute. Esophageal pressure is measuredusing a balloon catheter inserted into the middle of the esophagus. Thetranspulmonary pressure is the difference in pressures between theendotracheal tube and the esophageal catheter. Airflow rates aremeasured with a sensitive pneumotachograph, and the airflow resistanceis calculated by a method of electrical subtraction.

Tachykinins such as substance P are delivered by aerosol, intratrachealinstillation, or intravenously. A dose of mediator peptide thatincreases airflow resistance by approximately two times is used forsubsequent studies of airway responsiveness. A comparison of theresponse to the peptide agonist in the absence and presence of differentdoses of enkephalinase is made.

EXAMPLE 15 Measurement of Inflammatory Cell Responses In Vivo

Neutrophil chemotaxis, presence of eosinophils and other inflammatorycells, and mast cell degranulation in vivo, is measured in biopsyspecimens from airway and other tissues. Tissues are taken from controlanimals, animals treated with substance P or other endogenous peptidemediators, and from animals pretreated with enkephalinase before orafter exposing animals to endogenous peptides. Specimens are fixed in10% buffered formalin, imbedded in paraffin, and 3 sections 4 mm thickare obtained from each tissue. Sections are stained withhematoxylin-eosin followed by naphthol AS-D chloroacetate esterase. Thenumber of neutrophils or other cells are determined for each sectionfrom each biopsy specimen to assess airway inflammation. Cell counts aremade at 630×. The volume of epithelium or other tissue is determinedusing a digitizer (Model 614B; Talos Inc.) to obtain the area and thethickness of the section (4 mm), and data are expressed as the number ofcells per volume of tissue.

EXAMPLE 16 Effects of Enkephalinase on Esophageal Smooth MuscleContraction In Vivo

The contraction of the lower esophageal sphincter in anesthetizedanimals is measured using modifications of the methods publishedpreviously (Reynolds et al., Am. J. Physiol. [GastrointestinalPhysiology] 246: 346 [1984]). A catheter system consisting of two tubes,one for recording pressure and infusion of fluid or drugs, and anotherfor recovery of the fluid in the esophagus is inserted into theesophagus, and water is pumped (flow rate: approximately 0.75 ml/min)through the catheter. The fluid is continuously withdrawn from theesophagus so as to keep the volume of fluid in the esophagus constant,and the esophagus patent. By fixing the lower end of the esophagus toprevent shortening of the longitudinal muscles only circular muscularcontractions are recorded The pressure recording catheter is attached toa transducer (e.g., Statham Model P23) and pressure is recorded on achart recorder.

EXAMPLE 17 Effect of Enkephalinase on Renal Hypertension in Rats

Elevated arterial pressure or hypertension may be caused by renaldisease. One type of renal hypertension is due to activation of therenin-angiotensin system. Renin, a proteolytic enzyme produced by thekidney, converts angiotensinogen to the decapeptide, angiotensin I,which in turn is converted to angiotensin II. Angiotensin II is a potentpressor compound and exerts this pressor effect directly on arteriolarsmooth muscle. Enkephalinase cleaves angiotensin I, thus preventing itsconversion to angiotensin II and thus is likely to be an effectivetherapeutic in the treatment of renal hypertension.

The effects of enkephalinase on renal hypertension is studied using thefollowing procedure; one renal artery is narrowed producing highconcentrations of renin followed by high levels of angiotensinmanifested in systemic hypertension.

Systemic blood pressure is monitored by surgically placing a catheter ina femoral artery and bringing the catheter out of the body via the back.This allows the accurate monitoring of blood pressure in unanesthetizedand unrestrained animals. The effects of enkephalinase and enkephalinaseinhibitors on the arterial blood pressure, the blood levels of renin,angiotensin I and angiotensin II are monitored

EXAMPLE 18 Effect of Enkephalinase on Substance P In the Eye

Substance P-like biological activity or immunoreactivity has been foundin various parts of the eye, including the cornea and limbus, iris,ciliary body, and choroid. (Stone, R. A. and Kuwayama, Y., Arch.Ophthalmol. 103:1207 [1985]). Substance P-like activity has beenobserved in the retina of various animals including the chicken, pigeon,rat, guinea pig, rabbit, dog, cow and monkey (e.g. Stjernschantz, J. etal. J. Neurochemistry 38: 1323-1328 [1982]; Unger, W. G. et al., Exp.Eye Res. 19:367-377 [1981]).

The function of substance P in the eye is not established. It has beensuggested that substance P may mediate antidromic vasodilation andneurogenic plasma extravasation as part of the inflammatory response totrauma in the eye (Holmdahl, G. et al. Science 214:1029 [1981]).

The role of substance P in the retina, iridial smooth muscles, ocularcirculation and the blood-aqueous barrier, intraocular pressure,formation and outflow of aqueous humor has been reviewed.(Stjernschantz. J. in Pharmacology of the Eye ed. Sears, M. L.[Springer-Verlag, 1979]). Substance P has been shown to have markedeffects on the sphincter muscle of the iris (Mandahl, A. and Bill. A.Acta. Physiol. Scand. 109:26 [1980]). It has also been shown that thecontractile response to nerve stimulation of the rabbit iris sphinctercould be antagonized by a substance P analog. These results suggest thatsubstance P or a closely related peptide is specifically involved inthis response. (Leander, J. Acta Physiol. Scand 112:185-193 [1981]). Invivo a pupillary block and increased intra-ophthalmic pressureaccompanying intense and sustained miosis has been observed followingacute injury. (Al-Chadyan, A et al., Invest. Ophthalmol. Vis. Sci. 18:361-365 [1979]; Stjernschantz, J. et al., Invest. Ophthalmol. Vis. Sci.20:53-60 [1981]). Thus substance P may be involved in the sustained andintense miosis observed during cataract surgery or in response to acuteinjury to the eye.

Injection of high doses (≧1 μg) of substance P into the anterior chamberof the eye induced an increase in intraophthalmic pressure. (Mandahl. A.and Bill, A., Acta Physiol. Scand. 112:331-338 [1981]; Stjernschantz, J.et al., Invest. Ophthamol. Vis. Sci. 20:53-60 [1981]). Low doses (≦10ng) induce mioses but no appreciable increase in intraophthalmicpressure. (Nishiyana, A. et al., Jpn. J. Ophthal. 25:362-369 [1981]).

Since enkephalinase cleaves substance P, administration of enkephalinaseeither prior to, during or immediately following cataract surgery islikely to prevent the intense miosis and increased ophthalmic pressure.The effects of enkephalinase on the intense miosis and increasedophthalmic pressure may be studied using various opthalmic models inrabbits such as the aphakic rabbit model for studying the effects oftopically applied drugs (Mirate, D. J. et al., Curr.Eye Res.1[8]:491-493 [1981] and Anderson. J. A. et al., Arch. Opthalmol. 100[4]:642-645 [1982]). Contraction of the pupil and of the iris sphinctermuscle as well as intraocular pressure and drug absorption to areas ofthe eye is monitored during surgery with and without administration ofenkephalinase.

We claim:
 1. A method of treatment of disorders associated withsubstance P susceptible to cleavage by enkephalinase comprisingadministration of a therapeutically effective dose of enkephalinase toan animal affected by such disorder.
 2. The method of treatment of claim1 wherein the disorder is inflammation.
 3. The method of treatment ofclaim 1 wherein the disorder is airway disease.
 4. The method oftreatment of claim 1 wherein the disorder is increased ophthalmicpressure.
 5. The method of treatment of claim 3 wherein the airwaydisease is asthma.
 6. The method of treatment of claim 1 wherein thedisorder is a cough.
 7. The method of claim 1 wherein the enkephalinaselacks the cytoplasmic and transmembrane domain.
 8. The method of claim 1wherein the enkephalinase lacks the transmembrane domain.
 9. The methodof claim 1 wherein the enkephalinase lacks the cytoplasmic domain. 10.The method of claim 1 wherein the enkephalinase is unaccompanied byassociated native glycosylation.
 11. The method of claim 1 wherein theenkephalinase is administered intramuscularly.
 12. The method of claim 7wherein the enkephalinase is administered intravenously.
 13. The methodof claim 9 wherein the enkephalinase is administered by intrapulmonaryinhalation.
 14. The method of claim 1 wherein the enkephalinase isadministered topically.
 15. The method of claim 1 wherein theenkephalinase is administered in a therapeutically effective dose offrom about 0.01 mg/kg to about 25 mg/kg.
 16. The method of claim 1wherein the enkephalinase is administered in a therapeutically effectivedose of from about 0.1 mg/kg to about 20 mg/kg.
 17. The method oftreatment of claim 2 wherein the inflammation is allergic dermatitis.18. The method of claim 1 wherein the condition is contraction of irissphincter muscles.